Porcine G-CSF variants and their uses

ABSTRACT

The present invention relates to variants of porcine granulocyte colony stimulating factor (pG-CSF). The pG-CSF variants are useful in treating preventing or reducing the incidence of bacterial infections in swine. Methods of treating swine are disclosed.

This application is a national filing of PCT/US18/055203 filed Oct. 10,2018, and published in English as WO2019/075053, which claims thebenefit of priority to U.S. Provisional patent application Ser. No.62/570,877, filed Oct. 11, 2017, each of which is incorporated herein byreference in its entirety.

The present invention relates to variant porcine granulocyte colonystimulating factor (pG-CSF) polypeptides containing a synthetic aminoacid. The synthetic amino acid is modified by attachment of apoly(ethylene glycol) (PEG) molecule. The PEGylated pG-CSF variant isused to treat bacterial infections in a porcine. When the porcine is aperiparturient sow, the bacterial infection may be mastitis, metritis,and agalactia (MMA) syndrome. Reducing bacterial infections in pregnantsows improves piglet survival.

The economic impact of infectious diseases in food animal production iswell documented. Infectious diseases reduce profits, increase productioncosts, and endanger food products, as well as affect the performance,health, and welfare of the animal. Diseases can cause morbidity andmortality of newborn, young (e.g., replacement stock) or adult animals,resulting in devastating effects on food animal production.

In porcines, one such disease may be mastitis, metritis, and agalactia(MMA) syndrome. Mastitis is a bacterial infection of the udder. Only oneor two glands may be affected, or the infection could spread to multipleglands. Metritis is a bacterial infection of the urogenital tract,sometimes presented as vulval discharges. Agalactica is a reduction in,or the total loss of, milk production by a sow. MMA syndrome can behighly variable and may not present all the above symptoms. Thus, MMAsyndrome can be difficult to detect and diagnose, and it may not bedetected until a nursing piglet shows signs of hunger, weight loss, oreven death.

Because MMA syndrome is difficult to detect, a prophylactic treatment ispreferable. Use of antibiotics, particularly shared class antibiotics,is discouraged in food-producing animals, so a non-antibiotic therapy ispreferable in the treatment of porcines. A cytokine such as pG-CSF couldincrease neutrophil numbers in an animal, thus priming the innate immunesystem to respond quickly to a bacterial infection. Modifying a pG-CSFvariant with PEG could prolong the pharmokinetics and stability of thecytokine, thus potentiating its effects.

A PEGylated bovine G-CSF can be used to treat mastitis in dairy cattle(WO2010/011735). PEGylated human G-CSF has also been described(WO2000/044785). A PEGylated wild type porcine G-CSF has been proposedfor the treatment of respiratory infections, such as viral infections(WO2005/025593). As disclosed in various aspects herein, a PEGylatedpG-CSF variant elevates porcine blood neutrophil numbers, reduces theincidence of MMA syndrome in periparturient sows, and/or improves pigletsurvival.

The present invention provides porcine granulocyte colony stimulatingfactor (pG-CSF) variants having a consensus sequence of:X₁PLSPASSLPQSFLLKX₂LEQVRKIQADGAELQERLCATHKLCX₃PQELVLLGHSLGLPQASLSSCSSQALQLTGCLNQLHGGLVLYQGLLQALAGISPELAPALDILQLDVTDLATNIWLQX₄EDLRX₄APASLPTQGTVPTFTSAFQRRAGGVLVVSQLQSFLELAYRVLRYL AEP (SEQ IDNO: 13). The variable X₁ can be the dipeptide methionine alanine as inSEQ ID NOs: 2 and 9, the dipeptide norleucine alanine as in SEQ ID NOs:7and 10, alanine as in SEQ ID NOs:4 and 11, or absent as in SEQ ID NOs: 8and 12. The variable X₂ can be cysteine as in SEQ ID NOs: 2, 4, 7, and8, or X₂ can be serine as in SEQ ID NOs: 9, 10, 11, and 12. The variableX₃ can be a synthetic amino acid. The synthetic amino acid can bepresent at position 43 (position relative to the mature wild-type pG-CSFas given in SEQ ID NO: 3) as shown in SEQ ID NO: 13. The synthetic aminoacid can be para-acetyl phenylalanine (pAF). The variable X₄ can bemethionine as in SEQ ID NOs:2, 4, 8, 9, 11, and 12, or X₄ can benorleucine as in SEQ ID NOs:7 and 10. The pAF synthetic amino acid canbe covalently attached to a poly(ethylene glycol) (PEG) molecule. ThePEG can have a molecular weight of about 20 kD to about 50 kD, or amolecular weight of about 30 kD. Preferably, the PEG is a linear PEGmolecule.

The present invention provides a porcine granulocyte colony stimulatingfactor (pG-CSF) variant having a sequence of:MAPLSPASSLPQSFLLKCLEQVRKIQADGAELQERLCATHKLC[pAF]PQELVLLGHSLGLPQASLSSCSSQALQLTGCLNQLHGGLVLYQGLLQALAGISPELAPALDILQLDVTDLATNIWLQMEDLRMAPASLPTQGTVPTFTSAFQRRAGGVLVVSQLQSFLELAYRVLR YLAEP (SEQ IDNO: 2), wherein a para-acetyl phenylalanine (pAF) synthetic amino acidpresent at position 43 is covalently attached to a 30 kD linear PEGmolecule.

The present invention provides a pharmaceutical composition comprisingany of the pG-CSF variants described herein, and at least onepharmaceutically acceptable carrier, diluent, or excipient.

The present invention provides a method for treating a bacterialinfection in a porcine comprising administering a therapeuticallyeffective amount of any of the pG-CSF variants described herein to theporcine in need thereof. The bacterial infection may be mastitis,metritis and agalactia (MMA) syndrome. The porcine in need of treatmentmay be a periparturient sow. The therapeutically effective amount of apG-CSF variant may be about 10-100 μg/kg animal weight, or about 30-50μg/kg animal weight, or about 40 μg/kg animal weight. Administration ofany of the pG-CSF variants described herein may occur at least once 7days or less prior to farrowing (i.e. on or after day 107 of gestation),or at farrowing. In some aspects, the method of treating a bacterialinfection in a porcine may comprise a second administration of any ofthe pG-CSF variants described herein within 14 days or less afterfarrowing (i.e., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 orwithin 14 days after farrowing).

The present invention provides a method for stimulating innate immuneresponse in a porcine comprising administering a therapeuticallyeffective amount of any of the pG-CSF variants described herein to theporcine in need thereof. The porcine in need of stimulation may be aperiparturient sow. In other aspects the porcine in need of stimulationmay be a porcine with a compromised or weakened immune system. Infurther aspects the porcine in need of stimulation may be a porcine thathas or is at risk of developing an infection including, for example abacterial infection or a viral infection. In some aspects, the methodcan stimulate production of cytokines (e.g., interferons (IFNs), tumornecrosis factors (TNFs), colony stimulating factors (CSFs), and/orinterleukins (ILs)), and/or activate or increase levels of immune cellssuch as dendritic cells (DCs), lymphocytes (e.g., B cells, T cells, andnatural killer (NK) cells), and myelocytes (e.g., Mast cells,myeloblasts (e.g., basophils, eosinophils, neutrophils, monocytes, andmacrophages)). In some embodiments of these aspects, the method canstimulate or enhance neutrophil antibacterial function by, for example,increasing neutrophil myeloperoxidase-hydrogen peroxide-halide mediatedantibacterial function.

In methods that stimulate the innate immune response, thetherapeutically effective amount of a pG-CSF variant may be about 10-100μg/kg animal weight, or about 30-50 μg/kg animal weight, or about 40μg/kg animal weight. Administration of any of the pG-CSF variantsdescribed herein may occur at least once 7 days or less prior tofarrowing (i.e. on or after day 107 of gestation), or at farrowing. Insome aspects, the method of treating a bacterial infection in a porcinemay comprise a second administration of any of the pG-CSF variantsdescribed herein within 14 days or less after farrowing (i.e., within 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or within 14 days afterfarrowing).

The present invention provides a method for reducing piglet mortalitycomprising administering a therapeutically effective amount of any ofthe pG-CSF variants described herein to a periparturient sow. Thetherapeutically effective amount of a pG-CSF variant may be about 10-100μg/kg animal weight, or about 30-50 μg/kg animal weight, or about 40μg/kg animal weight. The administration of any of the pG-CSF variantsdescribed herein may occur at least once 7 days or less prior tofarrowing (i.e. on or after day 107 of gestation), or at farrowing. Insome aspects, the method of reducing piglet mortality may comprise asecond administration to the periparturient sow of any of the pG-CSFvariants described herein within 14 days or less after farrowing (i.e.,within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or within 14 days afterfarrowing).

The present invention provides for use of any of the pG-CSF variantsdescribed herein in the manufacture of a medicament for a bacterialinfection in a porcine. The bacterial infection may be MMA syndrome. Theporcine may be a periparturient sow. The present invention provides foruse of any of the pG-CSF variants described herein in the manufacture ofa medicament for reducing piglet mortality.

The present invention provides any of the pG-CSF variants describedherein for use in therapy. The therapy may be the treatment of asyndrome of bacterial infection in a porcine. The bacterial infectionmay be MMA syndrome. The porcine may be a periparturient sow. Thetherapy may be the reduction of piglet mortality. The pG-CSF variant maybe administered to a periparturient sow at about 10-100 μg/kg animalweight, or about 30-50 μg/kg animal weight, or about 40 μg/kg animalweight. The pG-CSF variant may be administered at least once 7 days orless prior to farrowing (i.e. on or after day 107 of gestation), or atfarrowing. A second administration may be given to the periparturientsow 14 days or less after farrowing.

A “bacterial infection” is the growth of one or more bacterial specieson or within the skin, mucus membranes, glands, eyes, ears, urogenitaltract, digestive tract, lungs, blood or organs of an animal. Thebacteria may be a species classified within the genera of Achromobacter,Actinobacillus, Actinomyces, Bacillus, Bordatella, Brucella,Clostridium, Corynebacterium, Erysipelothrix, Escherichia, Haemophilus,Klebsiella, Leptospira, Listeria, Mycoplasma, Pasteurella, Proteus,Pseudomonas, Salmonella, Sphaerophorus, Staphylococcus, Streptococcus,or Vibrio.

The term “porcine” as used herein, refers to a pig, especially thedomestic pig (Sus scrofa domesticus or Sus domesticus) and can includeminiature pigs as well as those breeds raised for meat production. By“pig”, “swine” or “porcine” is meant to include all pig breeds.

The periparturient sow is defined as the pregnant female porcine withinthe last weeks of gestation through the first few weeks post-farrowing.Farrowing, or giving birth, typically occurs at about 114 days ofgestation. A periparturient sow could be a pregnant female porcine fromabout 100 days of gestation to about 14 days post-farrowing. A piglet isa porcine from birth to weaning at about 3 weeks of age.

A “synthetic amino acid” refers to an amino acid that is not one of the20 common amino acids or pyrrolysine or selenocysteine. Examples of suchsynthetic amino acids include, but are not limited to, para-acetylphenylalanine (pAF), acetylglucosaminyl-L-serine, andN-acetylglucosaminyl-L-threonine. Some synthetic amino acids and theirincorporation into polypeptides and subsequent modification aredescribed in WO2010/011735 and in WO2005/074650.

As used herein, the terms “treating”, “to treat”, or “treatment”,include restraining, slowing, stopping, reducing, ameliorating, orreversing the progression or severity of a symptom, disorder, condition,or disease. In some aspects the disclosure provides methods that arespecific to prophylactic treatment of a symptom, disorder, condition, ordisease that is not observed or detected in an animal which may be atrisk of developing one or more such symptom, disorder, condition, ordisease. In some aspects, a treatment will be applied therapeutically.

The term “effective amount” as used refers to that amount of the pG-C SFvariant being administered which will have the desired effect, such aspreventing, treating, or reducing a bacterial infection in a porcine.When the porcine is a periparturient sow, treating bacterial infectionsin the sow can improve survival of piglets. The effective amount mayvary with factors such as the weight of the sow.

By “administering” is meant the injection of a therapeutically effectiveamount of the compounds and compositions containing said compoundsdisclosed. For example without limitation, administration can beintramuscular (i.m) or subcutaneous (s.c.).

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent depending on the context in which it isused. As used herein, “about” is meant to encompass variations of ±10%,±5%, or ±1% (i.e., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or±1%).

The pG-CSF variants of the present invention may readily be produced ina variety of cells including mammalian cells, bacterial cells such as E.coli, Bacillus subtilis, or Pseudomonas fluorescence, and/or in fungalor yeast cells. The host cells can be cultured using techniques wellknown in the art. The vectors containing the polynucleotide sequences ofinterest (e.g., the variants of pG-CSF and expression control sequences)can be transferred into the host cell by well-known methods, which varydepending on the type of cellular host.

For example, the calcium chloride transformation method is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other eukaryotic host cells. Variousmethods of protein purification may be employed and such methods areknown in the art and described, for example, in Deutscher, Methods inEnzymology 182: 83-89 (1990) and Scopes, Protein Purification:Principles and Practice, 3rd Edition, Springer, NY (1994).

The PEGylated pG-CSF variants can be formulated according to knownmethods to prepare pharmaceutically useful compositions. In some aspectsa formulation is a stable lyophilized product that is reconstituted withan appropriate diluent or an aqueous solution of high purity withoptional pharmaceutically acceptable carriers, preservatives, excipientsor stabilizers (see Remington, The Science and Practice of Pharmacy,19th ed., Gennaro, ed., Mack Publishing Co., Easton, Pa. 1995).

The PEGylated pG-CSF variant may be formulated with a pharmaceuticallyacceptable buffer, and the pH adjusted to provide acceptable stability,and a pH acceptable for administration. In some non-limiting examples, apH acceptable for administration may be in a range from about 5 to about8 (i.e., about 5, about 6, about 7, or about 8). Moreover, the PEGylatedpG-CSF compositions of the present invention may be placed into acontainer such as a vial, a cartridge, a pen delivery device, a syringe,intravenous administration tubing or an intravenous administration bag.

The following experimental examples are illustrative of pG-CSF variants,and their use in treating bacterial infections in swine, such as MMAsyndrome in a periparturient sow. Reducing the incidence of bacterialinfections in the sow also reduces piglet mortality. It will beappreciated that other embodiments and uses will be apparent to thoseskilled in the art and that the invention is not limited to thesespecific illustrative examples or preferred embodiments.

EXAMPLE 1

Different variants of the porcine granulocyte colony stimulating factor(pG-CSF) cDNA (GenBank Accession number U68481.1) are generated byintroducing a TAG stop codon in the selected positions by site-directedPCR mutagenesis. Also, the portion of the cDNA encoding the signalsequence is replaced by a single methionine codon (i.e. ATG). Forexample, a cDNA encoding wild type mature pG-CSF with the altered signalsequence could be:

-   -   1 atggcccctc tcagccctgc cagctccctg ccccagagct tcctgctcaa        gtgcttagag    -   61 caagtgagga aaatccaggc tgatggcgcc gagctgcagg agaggctgtg        tgccacccac    -   121 aagctgtgcc acccccagga gctggtgctg ctcgggcact ctctgggcct        cccccaggct    -   181 tccctgagca gctgctccag ccaggccctg cagctgactg gctgcctgaa        ccaactgcat    -   241 ggcggcctcg tcctctacca gggcctcctg caggccctgg cgggcatctc        cccagagctg    -   301 gcccccgccc tggacatact gcagctggat gtcaccgact tagccaccaa        catctggctg    -   361 cagatggaag acctgaggat ggccccggcc tcgcttccca cccagggcac        cgtgccgacc    -   421 ttcacctcgg ccttccagcg ccgggcagga ggggtcctgg ttgtctccca        gctgcagagc    -   481 ttcctggagc tggcgtaccg tgtcctgcgc tacctcgccg agccctga        (SEQ ID NO: 5). This variant would encode a polypeptide of:    -   1 MAPLSPASSL PQSFLLKCLE QVRKIQADGA ELQERLCATH KLCHPQELVL        LGHSLGLPQA    -   61 SLSSCSSQAL QLTGCLNQLH GGLVLYQGLL QALAGISPEL APALDILQLD        VTDLATNIWL    -   121 QMEDLRMAPA SLPTQGTVPT FTSAFQRRAG GVLVVSQLQS FLELAYRVLR YLAEP        (SEQ ID NO: 1). If the TAG stop codon is desired to replace the        H43 residue, the cDNA sequence could be:    -   1 atggcccctc tcagccctgc cagctccctg ccccagagct tcctgctcaa        gtgcttagag    -   61 caagtgagga aaatccaggc tgatggcgcc gagctgcagg agaggctgtg        tgccacccac    -   121 aagctgtgct agccccagga gctggtgctg ctcgggcact ctctgggcct        cccccaggct    -   181 tccctgagca gctgctccag ccaggccctg cagctgactg gctgcctgaa        ccaactgcat    -   241 ggcggcctcg tcctctacca gggcctcctg caggccctgg cgggcatctc        cccagagctg    -   301 gcccccgccc tggacatact gcagctggat gtcaccgact tagccaccaa        catctggctg    -   361 cagatggaag acctgaggat ggccccggcc tcgcttccca cccagggcac        cgtgccgacc    -   421 ttcacctcgg ccttccagcg ccgggcagga ggggtcctgg ttgtctccca        gctgcagagc    -   481 ttcctggagc tggcgtaccg tgtcctgcgc tacctcgccg agccctga        (SEQ ID NO: 6). The resulting polypeptide would be:    -   1 MAPLSPASSL PQSFLLKCLE QVRKIQADGA ELQERLCATH KLCxPQELVL        LGHSLGLPQA    -   61 SLSSCSSQAL QLTGCLNQLH GGLVLYQGLL QALAGISPEL APALDILQLD        VTDLATNIWL    -   121 QMEDLRMAPA SLPTQGTVPT FTSAFQRRAG GVLVVSQLQS FLELAYRVLR YLAEP        (SEQ ID NO: 2), where the “x” indicates the H43 residue replaced        with a synthetic amino acid, as described below.

Plasmids encoding the variants are transformed into E. coli cellscontaining the expanded genetic code system components for incorporationof the synthetic amino acid para-acetyl phenylalanine (pAF). Transformedcells are grown in media supplemented with pAF and induced to expresspG-CSF with pAF incorporated into the sites indicated. The expressionsystem has been described, for example, in WO 2010/011735 (incorporatedherein by reference), and is generally known in the art.

Expression of the transfected cDNA variants is induced with arabinose,the cells are harvested, and the target pG-CSF pAF site variants areisolated and purified by reverse phase high-pressure liquidchromatography (RP-HPLC). An activated 30 kD linear aminooxy-PEG issite-specifically conjugated to the incorporated pAF. PEG-pG-CSFconjugates are purified from excess PEG and unconjugated pG-CSF variantsby chromatography.

EXAMPLE 2

The in vitro biological activity of PEGylated pG-CSF (PEG-pG-CSF)variants is measured by the ability of the variants to induceproliferation of M-NSF-60 cells (ATCC CRL-1838). The concentration ofthe variants able to effect 50% of maximal proliferation (EC₅₀) isdetermined by comparison to a standard curve generated with wild type(WT) pG-CSF. Based on the results of expression as presented in Example1 and the biological assays described here, PEG-pG-CSF variants areselected for further study.

The in vivo activity of the selected candidate PEG-pG-CSF variants istested in a rodent model. Sprague Dawley rats (3/group) are treated with0.25 mg/kg body weight with a PEG-pG-CSF variant. Blood samples aretaken at 0 (pre-dosing), 1, 3, 6, 24, 48, 56, 72, 96, 144, 192, and 264hours for pharmokinetic (PK) analysis, and samples are taken at 24, 48,72, and 96 hours for a complete blood count (CBC analysis). The primarymeasurement in the CBC analysis is the number of neutrophils present. AH43 variant stimulates a higher level of neutrophil development thanother variants tested. All variants have similar PK profiles.

EXAMPLE 3

The PEG-pG-CSF H43pAF is prepared as follows. As in Example 1,expression of the transfected cDNA variants is induced with arabinose,the cells are harvested, and the pG-CSF H43pAF site variant is isolated,denatured and refolded, and purified by cation exchange liquidchromatography (CEX), using CAPTO Adhere Impres (GE HealthcareLifesciences). Briefly, the unpegylated pG-CSF H43pAF variant is loadedonto the column to a concentration of 1-5 mg/mL resin. The column iswashed with five column volumes (CV) 30 mM sodium acetate at pH 4.5.Elution of the pG-CSF H43pAF variant is with a linear gradient ofelution buffer (30 mM sodium acetate, 0.5 M NaCl, Ph 4.5), by washingwith 0-100% elution buffer over 20 CV.

Based on mass spectroscopy (MS) analysis of pG-CSF H43pAF, the isolatedpeptides include a main peak represented by SEQ ID NO: 2 and severaldifferent contaminants. The contaminants include loss of the N-terminalmethione (SEQ ID NO: 4), loss of both the N-terminal methionine andalanine (SEQ ID NO: 8), and substitution of norleucine for theN-terminal methionine (SEQ ID NO: 7). Norleucine is known to bemisincorporated instead of the amino acid methionine in high densityfermentation with E. coli. Norleucine incorporation is reduced by usingone or more of the following steps: feeding the fermentation solutionswith methionine; fermenting with complex media instead of defined media(the complex media has one or more non-defined components in itincluding but limited to glycerol, salts, amino acids, vitamins, yeastextracts, plant and animal hydrolysates, peptones, and tryptones);and/or lowering the temperature of the fermentation reaction mixturepost induction.

The pG-CSF H43pAF variant is taken from the cation exchangechromatography pool after using Capto SP Impres chromatography andbuffer exchanged into 30 mM sodium acetate, 4% sucrose, pH 4.0 using a10 kDa MWCO tangential flow filtration cassette. The pG-CSF H43pAFvariant is then concentrated to about 8.0 mg/mL using an Amicon Ultracentrifugal filter according to manufacturer's instructions. Onceconcentrated, 30K linear PEG (PEG can be purchased commercially from NOFAmerica Corporation or EMD Merck, for example) is added in a 6:1 molarratio of PEG to pG-CSF H43pAF variant. The PEG/pG-CSF variant mixture isthen incubated at about 28° C. for at least 21 hours. This methodresults in >98% of the pG-CSF variant being conjugated with PEG. Thepegylated variant can then be purified by CEX as above. When tested inthe M-NSF-60 cell bioassay (Example 2), the PEG-pG-CSF H43pAF varianthas an EC₅₀ of at least 0.40 ng/mL, demonstrating good binding andpotency characteristics.

Samples are frozen and thawed over five cycles by freezing at 0° C. in1.5 mL tubes and thawing in a room temperature water bath. Nosignificant impact is observed for the high molecular weight (HMW)protein profile over five cycles of freeze-thawing, demonstrating thestability of the variant in solution.

Two additional pG-CSF H43pAF variants are generated to attempt toimprove refolding efficiency and thermostability of the variant at 50°C. Cysteine 17 is changed to either alanine (C17A) or to serine (C17S,SEQ ID NOs: 9-12). These mutations do not improve refolding yield, butC17A has decreased thermostability. PEG-pG-CSF H43pAF/C17S does have aslightly improved EC₅₀ of 0.26 ng/mL.

EXAMPLE 4

The PEG-pG-CSF H43pAF variant is administered to sows to characterizechanges in blood neutrophils. Six sows of 1.5-5 years of age and anaverage body weight of 269.7 kg are given 40 μg/kg of the PEG-pG-CSFH43pAF variant by intramuscular injection on the side of the neck. ThePEG-pG-CSF H43pAF variant is suspended in 30 mM sodium citrate, 250 mMarginine, pH 6.0 at a concentration of 8.2 mg/mL. Animals do not receiveany concomitant medication following initiation of treatment. No adverseevents are observed.

Blood is taken on day 0 prior to dosing and on days 2, 7, 10, 14, 17 and21 post-dosing. Neutrophil counts are determined for each sow and a meanfor the treatment is determined for each day. Treatment with a singledose of the PEG-pG-CSF H43pAF variant results in measurable increases inblood neutrophil counts over a three-week period (Table 1). Additionaldoses would be expected to stimulate maintenance of the higherneutrophil levels.

TABLE 1 Effect of PEG-pG-CSF H43pAF variant on mean daily bloodneutrophil counts. Day 0 2 7 10 14 17 21 Neutrophils (1000/μL) 4.6543.68 25.08 30.1 18.53 15.14 9.83

EXAMPLE 5

The PEG-pG-CSF H43pAF variant is administered to periparturient sows tocharacterize the effect on mastitis, metritis, and agalactia (MMA)syndrome and on piglet survival.

Sows at 95-100 days of gestation are placed into farrowing crates andhygienic husbandry practices are followed until day 107 of gestation foreach sow. On day 107, blood is collected and then the sows (25 pergroup) are treated either with 40 μg/kg of the PEG-pG-CSF H43pAF variantas in Example 4 or with a sodium chloride solution as a negativecontrol. The sows are then placed in unhygienic conditions to stimulatedevelopment of MMA. The unhygienic conditions include placing a mat onthe grated floor of the farrowing crate to allow bedding and wastematerial to accumulate. Also, a mixture of water, feces, and pinesawdust (2:1:1) is used to contaminate the crates. No oxytocic orcorticosteroid drugs are given to the sows. Clinical observations andrectal temperatures are collected on each sow once daily in the morningbeginning on day 107 of gestation and continuing until a diagnosis ofMMA at which time the sow has all samples collected and is then removedfrom the study, given treatment, and has hygienic crate conditionsreturned. Samples collected include blood, rectal temperature, and swabsof infected glands.

Farrowing typically occurs on day 114 of gestation. Piglets are weighedand tagged within 12 hours of birth. Clinical observations of thepiglets are made twice daily, and weights are also measured on days 3,7, and 21 after birth.

Using the per protocol definition of disease, little difference wasobserved between the two groups. However, when vulvar discharge wasremoved from the definition of disease, the incidence of disease wasreduced by more than 50% in the PEG-pG-CSF treated group. The treatedgroup also had a greater number of piglets weaned compared to controlgroup (Table 2).

TABLE 2 Effect of PEG-pG-CSF on MMA incidence and piglet survival.Variable Control PEG-pG-CSF P value Sows (number/group) MMA 36% (9/25)32% (8/35) 1.0000 MMA excluding vulvar 28% (7/25) 12% (3/25) 0.2890discharge Piglets (Std. Error of Mean) Number born/litter 13.3 (0.78)13.1 (0.72) 0.8567 Number weaned/litter 8.3 (0.61) 9.4 (0.61) 0.2056Percent survival 65.3 (4.5) 72.4 (4.3) 0.2590 Range survival/litter0-100% 38-100% n/a Weaning weight 5.9 (0.21) 5.7 (0.19) 0.5944

What is claimed is:
 1. A porcine granulocyte colony stimulating factor(pG-CSF) variant consisting of a sequence of:X₁PLSPASSLPQSFLLKX₂LEQVRKIQADGAELQERLCATHKLC(pAF)PQELVLLGHSLGLPQASLSSCSSQALQLTGCLNQLHGGLVLYQGLLQALAGISPELAPALDILQLDVTDLATNIWLQX₃EDLRX₃APASLPTQGTVPTFTSAFQRRAGGVLVVSQLQSFLELAYRVLRYLAEP (SEQ ID NO:13); wherein X₁is selected from the group of methionine alanine,norleucine alanine, alanine only, and no amino acids; wherein X₂ iscysteine or serine; wherein X₃ is methionine or norleucine; and whereina para-acetyl phenylalanine (pAF) synthetic amino acid present atposition 43 is covalently attached to a poly(ethylene glycol) (PEG). 2.The pG-CSF variant of claim 1, wherein the PEG has a molecular weight ofabout 20 kD to about 50 kD.
 3. The pG-CSF variant of claim 1, whereinthe PEG has a molecular weight of about 30 kD.
 4. The pG-CSF variant ofclaim 1, wherein the PEG is linear.
 5. The pG-CSF variant of claim 1,wherein a para-acetyl phenylalanine (pAF) synthetic amino acid presentat position 43 is covalently attached to a 30 kD linear PEG.
 6. Apharmaceutical composition comprising the pG-CSF variant of claim 1, andat least one pharmaceutically acceptable carrier, diluent, or excipient.7. A method for treating MMA syndrome in a porcine comprisingadministering a therapeutically effective amount of the pGCSF variant ofclaim 1 to the porcine in need thereof.
 8. The method of claim 7,wherein the MMA syndrome comprises symptoms of mastitis, metritis and/oragalactia.
 9. The method of claim 7, wherein the porcine is aperiparturient sow.
 10. The method of claim 7, wherein thetherapeutically effective amount of pG-CSF is about 10-100 μg/kg animalweight.
 11. The method of claim 7, wherein the therapeutically effectiveamount of pG-CSF is about 30-50 μg/kg animal weight.
 12. The method ofclaim 7, wherein the administering occurs at least once within 7 daysprior to farrowing.
 13. The method of claim 7, wherein the administeringoccurs at farrowing.
 14. The method of claim 12, further comprising asecond administration no later than 14 days after farrowing.
 15. Amethod for increasing blood neutrophils in a porcine comprisingadministering a therapeutically effective amount of the pG-CSF variantof claim 1 to the porcine.
 16. The method of claim 15, wherein thetherapeutically effective amount of pGCSF is about 10-100 μg/kg animalweight.
 17. The method of claim 15, wherein the administering occurs atleast once within 7 days prior to farrowing.
 18. The method of claim 15,wherein the administering occurs at farrowing.
 19. The method of claim17, further comprising a second administration no later than 14 daysafter farrowing.
 20. A method for stimulating innate immune response byincreasing blood neutrophils in a porcine comprising administering atherapeutically effective amount of the pG-CSF variant of claim 1 to theporcine in need thereof.